Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and͞or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinasebased system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob͞ob (leptindeficient) mice. In contrast to evidence of liver toxicity, inf lammation, and cellular infiltration observed with Adleptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety͞toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications. Adenoviral (Ad) vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of current Ad vectors for many gene therapy applications is limited by the transient nature of transgene expression obtained by these vectors (1-7). Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression and the immunogenicity of these vectors, including ''leaky'' viral protein expression and the transgene that is delivered (8-15). The development of Ad vectors that are deleted in all viral protein-coding sequences offers the prospect of a potentially safer, less immunogenic vector with an insert capacity of up to 37 kb (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This vector is supplied in trans with the structural proteins required for packaging and rescue and is thus helper-dependent (HD) (24).Leptin has been recently identified as a potent modulator of weight and food intake. Daily delivery of recombinant leptin protein was shown to induce weight reduction, suppress appetite, and decrease blood insulin and glucose levels in ob͞ob (leptindeficient) mice (27)(28)(29). It has been shown that delivery of the leptin cDNA by first-generation Ad vectors (Ad-leptin) may substitute for daily recombinant leptin protein treatment, although the effects were transient in both lean and ob͞ob treated mice (7,30). In the present study, we delivered the leptin cDNA using the HD virus (HD-leptin), testing the hypothesis that elimination of the viral protein coding sequences would diminish the vector's cellular immunogenicity and toxicity, and hence support its longevity in vivo. Because both the viral proteins and the trans...

The interplay of translation and mRNA turnover has helped unveil how the regulation of gene expression is a continuum in which events that occur during the “birth” of a transcript in the nucleus can have profound effects on subsequent steps in the cytoplasm. Exemplifying this continuum is nonsense-mediated mRNA decay (NMD), the process wherein a premature stop codon affects both translation and mRNA decay. Studies of NMD helped lead us to the therapeutic concept of treating a subset of patients suffering from multiple genetic disorders due to nonsense mutations with a single small molecule drug that modulates the translation termination process at a premature nonsense codon. Here we review both translation termination and NMD, and our subsequent efforts over the last fifteen years that led to the identification, characterization, and clinical testing of ataluren, a new therapeutic with the potential to treat a broad range of genetic disorders due to nonsense mutations.

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